IONIZABLE LIPID CONTAINING BIODEGRADABLE ESTER BOND AND LIPID NANOPARTICLES COMPRISING SAME

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application under U.S.C. 371 of International Application No. PCT/KR2023/000742, filed Jan. 16, 2023, which claims priority to and the benefit of Korean Application No. KR 10-2022-0006692, filed Jan. 17, 2022, the contents of which are incorporated into the present application by reference in their entireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (PCTKR2023000742_Sequence listing.xml; Size: 6,973 bytes; and Date of Creation: Jan. 16, 2023) is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to a novel ionizable lipid containing a biodegradable ester bond. More particularly, the present disclosure relates to an ionizable lipid containing a biodegradable ester bond, lipid nanoparticles manufactured using the same, and use thereof.

BACKGROUND

A drug delivery system (DDS) is a technology engineered to deliver the necessary dosage of a drug effectively, minimizing side effects while maximizing efficacy and effectiveness. In particular, the conventional viral delivery systems have been proven to be effective as drug delivery vehicles in gene therapy, but several drawbacks such as immunogenicity, limitations in the size of injected DNA, and difficulties in mass production have limited the use of viruses as gene delivery systems.

Therefore, as an alternative to viral systems, a method of transporting nucleic acids into cells has been mainly used so far by mixing nucleic acids with positively charged lipids or polymers (named lipid-DNA conjugates (lipoplexes) and polymer-DNA conjugates (polyplexes), respectively). In particular, the lipid-DNA conjugates are widely used at the cellular level because of their ability to bind to nucleic acids and deliver the nucleic acids well into the cell, but in an in vivo environment, there are some disadvantages that the conjugates often cause inflammation in the body when injected locally, and when injected intravascularly, there is a disadvantage that the conjugates accumulate in tissues such as the lung, liver, and spleen, which are primarily first-pass organs.

In light of this background, the present inventors have made a diligent effort to develop a novel material capable of efficiently delivering anionic drugs, such as nucleic acids, to the target organ or cell with excellent drug encapsulation efficiency, and completed the present disclosure by confirming excellent drug delivery effects of a novel ionizable lipid containing a biodegradable ester bond of the present disclosure.

DISCLOSURE

Technical Problem

An object of the present disclosure is to provide an ionizable lipid containing a biodegradable ester bond having a novel structure, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

Another object of the present disclosure is to provide lipid nanoparticles comprising the ionizable lipid, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof.

Still another object of the present disclosure is to provide a composition for drug delivery comprising the lipid nanoparticles and an anionic drug.

Technical Solution

The present disclosure will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present disclosure may be applied to each of the other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, it cannot be considered that the scope of the present disclosure is limited by specific descriptions described below.

In order to achieve the above objects, the present inventors researched and made efforts, confirmed that an ionizable lipid containing an ester bond represented by the following Formula 1 is able to deliver drugs stably and effectively when manufactured into lipid nanoparticles, and have fewer side effects such as hepatotoxicity, and completed the present disclosure.

An Ionizable Lipid Containing Ester Bonds

An embodiment of the present disclosure for achieving the above object is an ionizable lipid represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

• • wherein A is

• • R₁ and R₂ are each independently any one selected from —H, —C_1-6alkyl, —C_1-6alkyl-NR₃R₄, or —C_2-12alkyl-(C═O)—Y— (CH₂)_xCH═CH—C_4-12alkyl, R₃ and R₄ are each independently any one selected from —H, —C_1-6alkyl, or —C_2-12alkyl-(C═O)—Y— (CH₂)_xCH═CH—C_4-12alkyl, R₅ is —C_1-6alkyl-NR₃R₄ or —C_2-12alkyl-(C═O)—Y— (CH₂) CH═CH—C_4-12alkyl,
  • Y is —O— or NR₆,
  • R₆ is —H or —C_1-3alkyl,
  • m is an integer from 0 to 5,
  • n is an integer from 1 to 11,
  • x is an integer from 1 to 5,
  • l is an integer from 2 to 10, and
  • p is an integer from 0 to 2.

The compound represented by Formula 1 above may be specifically defined as follows, but is not limited thereto:

• • A may be

• • R₁ and R₂ may be each independently any one selected from —H, —C_1-3alkyl, —C_1-4alkyl-NR₃R₄, or —C_4-8 alkyl-(C═O)—Y— (CH₂)_xCH═CH—C_4-8 alkyl, R₃ and R₄ may be each independently any one selected from —H, —C_1-3alkyl, or —C_4-8 alkyl-(C═O)—Y— (CH₂)_xCH═CH—C_4-8 alkyl,
  • Y may be —O—,
  • m may be an integer from 0 to 3,
  • n may be an integer from 3 to 7,
  • x may be an integer from 1 to 3,
  • l may be an integer from 2 to 6, and
  • p may be 1.

Further, the compound represented by Formula 1 above may be defined as follows, but is not limited thereto:

• • A may be

• • R₃ and R₄ may be each independently any one selected from —H, —C_1-3alkyl, or —C_4-8 alkyl-(C═O)—Y— (CH₂)_xCH═CH—C_4-8 alkyl,
  • R₅ may be —C_1-4alkyl-NR₃R₄ or —C_4-8 alkyl-(C═O)—Y— (CH₂)_xCH═CH—C_4-8 alkyl,
  • Y may be —O—,
  • m may be an integer from 0 to 3,
  • n may be an integer from 3 to 7,
  • x may be an integer from 1 to 3,
  • l may be an integer from 2 to 6, and
  • p may be 1.

Further, according to an embodiment of the present disclosure, the compound represented by Formula 1 above may be selected from the group consisting of the compounds listed in Table 1 below.

As used herein, the term “alkyl” refers to a straight-chain or branched-chain, non-cyclic saturated hydrocarbon, unless otherwise indicated. For example, “C_1-6alkyl” may refer to an alkyl containing from 1 to 6 carbon atoms. The addition of simple substituents in the alkyl structures of the present disclosure, for example, is all equally included in the scope of the present disclosure as long as they have an equivalent effect to the ionizable lipids of the present disclosure.

As used herein, the term “ionizable lipid” refers to an amine-containing lipid capable of being readily protonated, and is also referred to as a lipidoid. The ionizable lipid, the charge state of which may change depending on the ambient pH, plays a role in electrostatic interaction with anionic drugs to ensure that the drug is encapsulated in the lipid nanoparticle with high efficiency and contributes to the structure of the lipid nanoparticles. The ionizable lipid of the present disclosure is characterized by having a form in which an alkyl chain containing an ester bond and a double bond is bound to an amine head containing at least one primary amine, and has the advantage of having superior efficacy such as tissue specificity and gene expression rate in vivo compared to other known ionizable lipids in nucleic acid delivery, and is easily degraded in vivo post-drug delivery, resulting in minimal side effects such as hepatotoxicity.

As used herein, the term “stereoisomer” refers to compounds that have the same chemical or molecular formula but are sterically different. Each of these stereoisomers and mixtures thereof are also included within the scope of the present disclosure. Unless otherwise specified, a solid bond (−) connected to an asymmetric carbon atom may include a wedged bond () or wedge dashed bond () representing the absolute arrangement of stereocenters.

The compound represented by Formula 1 of the present disclosure may be present in the form of a “pharmaceutically acceptable salt”. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid may be useful, but is not limited thereto. The term “pharmaceutically acceptable salt” as used herein refers to any arbitrary organic or inorganic acid addition salt or base addition salt of the compound whose side effects do not reduce the beneficial efficacy of the compound represented by Formula 1 at concentrations having an effective action that is relatively non-toxic and harmless to a patient.

The acid addition salts may be prepared by conventional methods, for example, by dissolving a compound in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. An equimolar amount of the compound and an acid or alcohol in water may be heated, and then the mixture may be evaporated to dryness, or the precipitated salt may be suction filtered.

Here, organic acids and inorganic acids may be used as free acids. Examples of the inorganic acid may include, but are not limited to, hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid, and examples of the organic acid may include, but are not limited to, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, or hydroiodic acid.

Further, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal salt or alkaline earth metal salt may be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, and filtering an insoluble compound salt, followed by evaporating and drying the filtrate. Here, the metal salts may be, but are not limited to, sodium, potassium, or calcium salts. In addition, the corresponding silver salt may also be obtained by reacting alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

Lipid Nanoparticles Comprising an Ionizable Lipid Comprising Ester Bonds

Another aspect of the present disclosure is lipid nanoparticles comprising the ionizable lipid represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The lipid nanoparticles of the present disclosure may comprise one, or two or more ionizable lipids represented by Formula 1. In addition, depending on the purpose, the lipid nanoparticles of the present disclosure may further comprise ionizable lipids other than those of the present disclosure.

The lipid nanoparticles may further comprise at least any one selected from the group consisting of phospholipids, cholesterol, and PEG-lipids, but are not limited thereto.

The phospholipid serves to wrap and protect the core formed by the interaction of the ionizable lipid and drugs within the lipid nanoparticles, and bind to a phospholipid bilayer of the target cell to facilitate cell membrane penetration and endosomal escape during intracellular delivery of the drug. The phospholipid may be any phospholipid capable of promoting the fusion of lipid nanoparticles without limitation, and for example, may be dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate (18-PA), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, or a mixture thereof.

The structural lipid serves to impart rigidity to the lipid loading within the lipid nanoparticles in terms of morphology and to improve the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles. The structural lipid may be, for example, but not limited to, cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or a mixture thereof.

In the present disclosure, PEG-lipid refers to a form in which the lipid and PEG are conjugated, indicating that the lipid has a hydrophilic polymer, a polyethylene glycol polymer, bonded at one end. The PEG-lipid contributes to the particle stability of the nanoparticles in serum within the lipid nanoparticles and acts as a barrier to inter-nanoparticle aggregation. In addition, PEG-lipid is able to protect nucleic acids from degradative enzymes during in vivo delivery, thereby enhancing the in vivo stability of nucleic acids and increasing the half-life of drugs encapsulated in nanoparticles. The PEG-lipid may be, for example, but not limited to, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, or a mixture thereof.

As an embodiment, when lipid nanoparticles are manufactured by mixing the ionizable lipid of the present disclosure with phospholipid, structural lipid, and PEG-lipid, a molar ratio of ionizable lipid: phospholipid: structural lipid: PEG-lipid may be 10 to 40:10 to 30:40 to 70:1 to 5. Further, the molar ratio may be, but is not limited to, 20 to 30:15 to 25:45 to 55:1 to 5, but is not limited thereto.

The lipid nanoparticle of the present disclosure may have a pKa of 6.0 to 7.0, more specifically 6.5 to 7.0 to exhibit a positive charge under acidic pH conditions, and thus the lipid nanoparticles are able to easily form drug complexes through electrostatic interaction with therapeutic agents such as nucleic acids and anionic drugs, which exhibit a negative charge, thereby encapsulating the anionic drugs with high efficiency and being usable as intracellular or in vivo drug delivery composition. Thus, the lipid nanoparticles of the present disclosure may be useful for the delivery of not only nucleic acids, but also any anionic form of drug. In other words, the lipid nanoparticles of the present disclosure may be finally manufactured in a form that further comprises an anionic drug (in an enclosed form).

As used herein, the term “encapsulation” refers to a process of encapsulating a delivery substance for surrounding and embedding it in vivo efficiently, and the drug encapsulation efficiency (encapsulation efficiency) means the amount of drug encapsulated within lipid nanoparticles relative to the total drug amount used in the preparation.

The anionic drug may be a nucleic acid, a small molecule compound, a peptide, a protein, a protein-nucleic acid construct, or an anionic biopolymer-drug conjugate, but is not limited thereto, as long as the drug is able to be stably and efficiently delivered by forming the lipid nanoparticles with the ionizable lipid of the present disclosure.

In the present disclosure, the nucleic acid may be, but is not limited to, siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme, or a mixture thereof.

A weight ratio of the ionizable lipid/nucleic acid in the lipid nanoparticle may be 1 to 20, such as 5 to 15, more specifically 8 to 12, but is not limited thereto.

In the present disclosure, the lipid nanoparticle may have a diameter size of, for example, 50 to 90 nm, but is not limited thereto.

Composition for Drug Delivery and Pharmaceutical Composition Comprising Lipid Nanoparticles

Still another aspect of the present disclosure is a composition for drug delivery, comprising anionic drug-containing lipid nanoparticles according to the present disclosure.

Still another aspect of the present disclosure is a pharmaceutical composition comprising anionic drug-containing lipid nanoparticles according to the present disclosure as an active ingredient.

The lipid nanoparticles and anionic drugs are described above.

The lipid nanoparticles of the present disclosure are effective for delivering anionic drugs because it is possible to form stable complexes with anionic drugs such as nucleic acids and exhibit low cytotoxicity and effective cellular uptake. Thus, the lipid nanoparticles have preventive or therapeutic effects on related diseases depending on the type of anionic drug used and the type of nucleic acid used, and have unlimited potential for utilization as compositions for drug delivery.

As used herein, the term “treatment” refers to intervention aimed at altering the natural processes of individuals or cells having a disease, which may be performed either during the progression of pathological conditions or for prevention thereof. The intended therapeutic effect includes preventing the onset or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathologic consequences of the disease, preventing metastasis, slowing the rate of disease progression, alleviating or temporarily relieving a disease condition, achieving remission, or improving prognosis. In particular, the present disclosure encompasses any act of ameliorating the course of a disease by the administration of lipid nanoparticles comprising ionizable lipid containing a disulfide bond, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and an anionic drug as an active ingredient. Further, the term “prevention” refers to any act of inhibiting or delaying the onset of a disease by the administration of the lipid nanoparticles. When the lipid nanoparticles of the present disclosure are used for treatment or prevention purposes, the lipid nanoparticles are administered to an individual in a therapeutically effective amount.

As used in the present disclosure, the term “therapeutically effective amount” refers to an effective amount of anionic drug-containing lipid nanoparticles. Specifically, the “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may be determined depending on factors including the subject type and severity, age, sex, type of diseases, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, or may be administered sequentially or simultaneously with a commercially available therapeutic agent. In addition, the pharmaceutical composition of the present disclosure may be administered singly or in multiple doses. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with the minimum amount without side effects, which may be easily determined by those skilled in the art. The administration dose of the pharmaceutical composition of the present disclosure may be determined by specialists according to various factors such as the patient's condition, age, sex, complications, and the like. Since the active ingredient of the pharmaceutical composition of the present disclosure has excellent safety, the active ingredient may be used even above the predetermined dose.

The compositions comprising the lipid nanoparticles as active ingredients may be administered by oral, intramuscular, intravenous, arterial, subcutaneous, peritoneal, pulmonary, and nasal injection, without limitation.

The composition of the present disclosure may further comprise one or more additional pharmaceutically acceptable carriers for administration. The pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and, if necessary, may contain other conventional additives such as antioxidants, buffers, bacteriostatic agents, and the like. Further, the composition may be formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, and the like, pills, capsules, granules or tablets by further adding diluents, dispersants, surfactants, binders and lubricants. Accordingly, the pharmaceutical composition of the present disclosure may be a patch, liquid, pill, capsule, granule, tablet, suppository, or the like. These preparations may be prepared by conventional methods used for formulation in the art or methods disclosed in the document [see, Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and formulated into various preparations depending on respective diseases or components.

Exemplary embodiments of the present disclosure may be modified in various other forms, and the scope of the present disclosure is not limited to the exemplary embodiments to be described below. In addition, the exemplary embodiments of the present disclosure are provided to more completely explain the present disclosure to an ordinary person skilled in the art. Further, “including” a component throughout the specification does not mean excluding other components, but rather it means that other components may be further included, unless otherwise stated.

Advantageous Effects

The ionizable lipid containing an ester bond of the present disclosure may be employed for the stable delivery of anionic drugs when manufactured into lipid nanoparticles, particularly effective in nucleic acid delivery, which may be useful in related technologies such as lipid nanoparticle-mediated gene therapy.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the pKa measurement of 244-9-cis lipid nanoparticles by the TNS experiment.

FIG. 2 shows the results of siFVII (Factor VII) knockout effect in an in vivo efficacy experiment, obtained by testing the hepatocyte targeting potential of siRNA-encapsulated lipid nanoparticles containing ionizable lipid of the present disclosure.

FIG. 3 shows hEPO levels analyzed in blood collected from mice after intravenous injection to confirm in vivo delivery of hEPO mRNA-encapsulated 244-9-cis lipid nanoparticles.

FIG. 4 is an image showing bioluminescence after intramuscular injection into mice to confirm in vivo delivery of mFLuc encapsulated 244-9-cis lipid nanoparticles.

FIG. 5 shows the results of cytotoxicity of 244-9-cis lipid nanoparticles obtained after manufacturing lipid nanoparticles without encapsulating nucleic acid drugs and then treating four cell lines, HepG2, MEF, Hela, and KB-GFP, with the lipid nanoparticles at different concentrations to confirm cytotoxicity thereof.

FIG. 6 shows the results of AST and ALT levels after preparation and administration mRNA-encapsulated lipid nanoparticles to mice in order to confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, as compared to ALC-0315 lipid.

FIG. 7 shows the results of ALP and ALB levels after preparation and administration of mRNA-encapsulated lipid nanoparticles in order to confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, as compared to ALC-0315 lipid.

FIG. 8 shows a liver histogram confirming the liver toxicity of 244-9-cis lipid nanoparticles 24 hours after mFLuc-encapsulated lipid nanoparticles were manufactured and administered to mice, as compared to ALC-0315.

BEST MODE

Herein after, the present disclosure will be described in more detail through Preparation Examples, Examples and Experimental Examples. However, the following Preparation Examples, Examples and Experimental Examples are provided only for illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

Example 1. Preparation of an Ionizable Lipid Containing Ester Bond

Example 1-1. Synthesis of an Ionizable Lipid Containing Ester Bond

An ionizable lipid containing an ester bond of the present disclosure was prepared by combining various kinds of amine headgroups with alkyl chains containing an ester bond and a double bond.

As to Compound 1 to Compound 4, specifically, 9-bromo nonanoic acid and N,N-diisopropylcarbodiimide (DIC) (each 1.5 eq.), and 4-dimethylaminopyridine (DMAP) (0.2 eq.) were added per cis-2-nonen-1-ol in dichloromethane (DCM) solvent and reacted overnight at 25° C., followed by purification in hexane/ethyl acetate (5:1 v/v ratio) using a CombiFlash column. After the solvent was blown off, each product was dissolved in ethanol. Then, N,N-diisopropylethylamine (DIPEA) (1 eq.) was added, followed by the addition of a substance containing primary amine (amine head) at 0.3 eq. (for Compound 1), 0.2 eq. (for Compounds 2 and 4), and 0.15 eq. (for Compound 3), and reacted for 3 days. Each reaction product was purified in DCM/MeOH (9:1 v/v ratio) using a CombiFlash column to synthesize the ionizable lipid.

As to Compound 5 to Compound 9, specifically, 9-bromo nonanoic acid for Compound 5, 5-bromo pentanoic acid for Compound 6, 6-bromo hexanoic acid for Compound 7, 7-bromo heptanoic acid for Compound 8, or 8-bromo octanoic acid for Compound 9, and N,N-diisopropylcarbodiimide (DIC) (each at 1.5 eq.), and 4-dimethylaminopyridine (DMAP) (0.2 eq.) were added per cis-2-nonen-1-ol in dichloromethane (DCM) solvent and reacted overnight at 25° C., followed by purification in hexane/ethyl acetate (5:1 v/v ratio) using a CombiFlash column. After the solvent was blown off, each product was dissolved in ethanol. Then, N,N-diisopropylethylamine (DIPEA) (1 eq.) and a substance containing primary amine (amine head) (0.3 eq.) were added, and reacted for 3 days. Each reaction product was purified in DCM/MeOH (9:1 v/v ratio) using a CombiFlash column to synthesize the ionizable lipid.

For example, the specific Reaction Scheme for 244-9-cis is shown in Reaction Scheme 1 below.

As to Compound 10 to Compound 11, specifically, 9-bromo nonanoic acid and N,N-diisopropylcarbodiimide (DIC) (each at 1.5 eq.), and 4-dimethylaminopyridine (DMAP) (0.2 eq.), were added per cis-3-nonen-1-ol for Compound 10, or cis-4-decen-1-ol for Compound 11 in dichloromethane (DCM) solvent and reacted overnight at 25° C., followed by purification in hexane/ethyl acetate (5:1 v/v ratio) using a CombiFlash column. After the solvent was blown off, each product was dissolved in ethanol. Then, N,N-diisopropylethylamine (DIPEA) (1 eq.) and a substance containing primary amine (amine head) (0.3 eq.) was added, and reacted for 3 days. Each reaction product was purified in DCM/MeOH (9:1 v/v ratio) using a CombiFlash column to synthesize the ionizable lipid.

Specific exemplary compounds of the ionizable lipids containing ester bonds of the present disclosure are shown in Table 2 below.

Example 1-2. Confirmation of Synthesis of the Ionizable Lipid Containing Ester Bonds

To confirm the synthesis of the ionizable lipids prepared in Example 1-1 above, MS analysis was performed. Specifically, the ionizable lipids were diluted in ethanol to a concentration of 0.5 ppm or less for MS analysis, wherein the instrument used for the analysis was a 6230 LC/MS from Agilent Technologies (Palo Alto, USA), and the separation column was Zorbax SB-C18 (100 mm×2.1 mm i.d., 3.5 μm) from Agilent Technologies. The MS analysis results are shown in Table 3 below.

It could be seen from the above result that the ionizable lipids containing ester bonds in Example 1-1 were successfully synthesized.

Example 2. Manufacture of Lipid Nanoparticle

Example 2-1. Preparation Parameter

Lipid nanoparticles containing the ionizable lipids of the present disclosure were manufactured at the weight ratios in Table 4 and Table 5 below.

Example 2-2. Manufacture of siRNA-Encapsulated Lipid Nanoparticles

Each ionizable lipid prepared in Example 1-1 above, cholesterol powder (BioReagent, suitable for cell culture, ≥99%, Sigma, Korea), phospholipid (DOPE) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol in a molar ratio of 26.5:20:52:1.5.

Subsequently, siRNA was dissolved in 50 mM sodium acetate buffer (Sigma, Korea). The above prepared ethanol containing each ionizable lipid, cholesterol, phospholipid, and lipid-PEG dissolved therein was mixed with the acetate buffer at a volume ratio of 1:3 through a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min, thereby manufacturing lipid nanoparticles.

Example 2-3. Manufacture of mRNA-Encapsulated Lipid Nanoparticles

The ionizable lipids prepared in Example 1-1 above, cholesterol powder (BioReagent, suitable for cell culture, ≥99%, Sigma, Korea), phospholipid (DOPE) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol in a molar ratio of 26.5:20:52:1.5.

Subsequently, mRNA was dissolved in 10 mM sodium citrate buffer (Sigma, Korea). The above prepared ethanol containing each ionizable lipid, cholesterol, phospholipid, and lipid-PEG dissolved therein were mixed with the citrate buffer at a volume ratio of 1:3 through a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min, thereby manufacturing lipid nanoparticles.

Experimental Example 1: Confirmation of Physicochemical Characterization of Lipid Nanoparticles

Experimental Example 1-1. Measurement of Particle Size, Polydispersity Index (PDI), Surface Charge (Zeta Potential) and Drug Encapsulation Efficiency

The physicochemical properties of lipid nanoparticles encapsulated with firefly luciferase mRNA (SEQ ID No: 1) or FVII siRNA (SEQ ID Nos: 2 and 3) manufactured in Example 2-3 above were to be measured in the present Experimental Example. Specifically, 244-9-cis lipid nanoparticles encapsulated with mRNA or siRNA were each manufactured and subsequently diluted with PBS to achieve a concentration of 1 μg/ml for the RNA within each lipid nanoparticle. Then, the diameter, polydispersity index (PDI), and surface charge (zeta potential) of the lipid nanoparticles were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

Next, the encapsulation efficiency (drug encapsulation efficiency, %) of lipid nanoparticles encapsulated with mRNA or siRNA above was determined by Ribogreen assay (Quant-iT™ RiboGreen® RNA, Invitrogen). The lipid nanoparticles encapsulated with mRNA were diluted with 50 μl of 1×TE buffer to a final concentration of 4 to 7 μg/ml of mRNA in 96-well plate. 50 μl of 1×TE buffer was added to the group without Triton-X treatment (Triton-x LNPs(−)), while 50 μl of 2% Triton-X buffer was added to the group treated with Triton-X (Triton-X LNPs(+)). After incubation at 370° C. for 10 minutes, the lipid nanoparticles were decomposed with Triton-X to release the encapsulated nucleic acids. Then, 100 μl of Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNPs (−) and Triton LNPs (+) was measured by wavelength bandwidth (excitation: 485 nm, emission: 528 nm) on an Infinite® 200 PRO NanoQuant (Tecan), and the drug encapsulation efficiency (%) was calculated as shown in Equation 1 below.

Drug ⁢ Encapsulation ⁢ Efficiency ⁢ ( % ) = ( Fluorescence ⁢ of ⁢ Triton ⁢ LNP ⁡ ( + ) - Fluorescence ⁢ of ⁢ Triton ⁢ LNP ⁡ ( - ) ) ⁠ / ( Fluorescence ⁢ of ⁢ Triton ⁢ LNP ⁡ ( + ) ) × 100 [ Equation ⁢ 1 ]

The results for each are as follows (Table 6).

Experimental Example 1-2. Measurement of pKa

In the present Experimental Example, the pKa of the firefly luciferase mRNA (SEQ ID NO: 1)-encapsulated lipid nanoparticles formulated in Example 2-3 above was calculated by in vitro TNS (2-(p-toluidino)naphthalene-6-sulfonic acid) assay.

Specifically, solutions containing 20 mM sodium phosphate, 25 mM citric acid, 20 mM ammonium acetate, and 150 mM sodium chloride were prepared at various pH levels by adjusting the pH of the solution using 0.1 N sodium hydroxide and/or 0.1 N hydrochloric acid in increments of 0.5, starting from pH 3.5 and ending at pH 11. Each (100 μl) of the solutions with varying pH was added to a black 96-well plate, and 300 μM of TNS stock solution was added to each of the above solutions to a final concentration of 6 μM. The formulated lipid nanoparticles were added to the mixed solution to a final concentration of 20 μM, and the fluorescence intensity was measured (excitation: 325 nm, emission: 435 nm) using a Tecan instrument. Here, the pKa for the lipid nanoparticles was calculated as the pH value at which half of the maximum fluorescence was reached.

As a result, it was found that the lipid nanoparticles of the present disclosure have a pKa value of about 6.2 and exhibit the shape of an s-shaped curve on the graph (FIG. 1). Anionic TNS interacts with positively charged ionizable lipids to become lipophilic, and as the pH value approaches the pKa value of each LNP, the lipophilicity of TNS decreases, leading to increased quenching of TNS fluorescence by more water molecules, and thus lipid nanoparticles with a pKa of 6.0 to 7.0 have good in vivo drug delivery efficiency. In addition, the lipid nanoparticles that exhibit an “s-shaped curve” in the graph of pH-dependent fluorescence indicate easy interaction with endosomal membrane and the ability to facilitate endosomal escape upon acidification.

Experimental Example 1-3. Cryo-TEM Measurement

Cryo-TEM was used to image the internal structure of the lipid nanoparticles.

Specifically, lipid nanoparticles encapsulated with firefly luciferase mRNA (SEQ ID NO: 1) were concentrated to a final concentration of 15 to 25 mg/ml of total lipid, and 2 to 4 μl of the LNP solution was loaded onto a copper grid and blotted. An internal image of the lipid nanoparticles was then measured using a cryo-TEM (FEI Tecnai F20 G2) instrument at the KIST Advanced Analysis Center.

Experimental Example 2: Confirmation of In Vitro Efficacy of mRNA-Encapsulated Lipid Nanoparticles

To determine the efficacy of mRNA-encapsulated lipid nanoparticles in vitro, screening was performed using lipid nanoparticles synthesized with various amine head groups.

The test was conducted by treating 20 ng of mFLuc (SEQ ID NO: 1)-encapsulated lipid nanoparticles into HeLa cells using each lipid nanoparticle synthesized with various amine headgroups (Compounds 1, 2, 4, and 5). The luminescence intensity was measured at 24 hours.

As a result, as shown in Table 7 below, a significant increase in luminescence intensity was observed for most of the lipid nanoparticles tested, and in particular, it could be seen that Compound 5 (2-(4-9-cis) lipid nanoparticles showed the highest expression effect.

Experimental Example 3: Confirmation of In Vivo Effects of Lipid Nanoparticles

Experimental Example 3-1. Delivery Effect of siRNA-Encapsulated Lipid Nanoparticles

Since FVII is specifically expressed in hepatocytes, the hepatocyte targeting potential of lipid nanoparticles was confirmed by Factor VII (FVII) knockout effect using siFVII.

Specifically, 244-9-cis lipid nanoparticles encapsulated with FVII-targeting siRNAs (SEQ ID NOs: 2 and 3) were manufactured by the method of Example 2-2 above. SiRNA-encapsulated 244-9-cis lipid nanoparticles manufactured at a concentration of 0.03, 0.1, or 0.3 mg/kg, based on the concentration of siRNA contained in the lipid nanoparticles, were injected intravenously into C₅₇BL/6 female 7-week-old mice. After 3 days, blood was collected and analyzed according to the protocol of the coaset FVII assay kit. The expression level of FVII was measured by plotting a standard curve with the blood from mice administered with PBS.

As a result, as shown in FIG. 2, the lipid nanoparticles manufactured using the ionizable lipids of the present disclosure effectively inhibited FVII expression in vivo in a concentration-dependent manner of the encapsulated siRNA, confirming that the lipid nanoparticles of the present disclosure could effectively deliver nucleic acids to target hepatocytes. In particular, lipid nanoparticles containing the ionizable lipids of the present disclosure showed superior FVII expression inhibition compared to lipid nanoparticles manufactured with FDA-approved ALC-0315 at all doses.

Experimental Example 3-2. Confirmation of Delivery Efficacy of mRNA-Encapsulated Lipid Nanoparticles Via Intravenous Injection

To investigate the mRNA delivery of the 244-9-cis lipid nanoparticles, luciferase mRNA was delivered into mice, and gene expression was confirmed by bioluminescence.

Specifically, 244-9-cis lipid nanoparticles encapsulated with mFluc (SEQ ID NO: 1) were manufactured by the method of Example 2-3 above. Then, 7-week-old C₅₇BL/6 mice were injected intravenously with the manufactured lipid nanoparticles (2 μg based on mRNA), and 3 hours later, administered intraperitoneally with luciferin 0.25 mg/kg, and bioluminescence was detected by IVIS (PerkinElmer, USA). The results showed that most of the lipid nanoparticles were delivered to the liver (Table 8).

Next, lipid nanoparticles encapsulated with hEPO mRNA (SEQ ID NO: 4) were manufactured in the same manner as above. The manufactured lipid nanoparticles at a dose of 0.1 mg/kg based on mRNA were injected intravenously into 7-week-old C₅₇BL/6 mice, and blood was collected after 3, 6, 9, 24, and 48 hours, respectively, for quantitative analysis of hEPO using the hEPO ELISA kit. As a result, the lipid nanoparticles containing the ionizable lipid of the present disclosure effectively delivered nucleic acids in vivo, resulting in significant levels of hEPO detection in the blood, which was confirmed by an approximately 1.7-fold higher the area under the curve compared to lipid nanoparticles manufactured with ALC-0315, the control (Table 9 and FIG. 3).

Experimental Example 3-3. Delivery Effect of mRNA-Encapsulated Lipid Nanoparticles Via Intramuscular Injection

Luciferase mRNA (SEQ ID NO: 1) was delivered to mice by intramuscular injection, and gene expression was confirmed by bioluminescence.

Specifically, mFluc-encapsulated lipid nanoparticles (2-(4-9-cis) were manufactured as described above, and 2 μg (based on mRNA) of the lipid nanoparticles were intramuscularly injected into 7-week-old C₅₇BL/6 mice. After 3 hours, luciferin 0.25 mg/kg was administered intraperitoneally and bioluminescence was confirmed by IVIS (PerkinElmer, USA).

The results showed that most of the lipid nanoparticles were well delivered to the injection site (Table 10 and FIG. 4).

Experimental Example 4: Determination of Cytotoxicity of Lipid Nanoparticles

The substance CCK-8 (tetrazolium salt) forms orange formazan through reduction by dehydrogenase in the mitochondria of living cells, so cell viability can be confirmed by absorbance analysis.

Specifically, different kinds of cells (HeLa, HepG2, MEF, KB-GFP) were seeded (0.4×10⁵) in transparent 96-well plates (SPL, 30096). Subsequently, the lipid nanoparticle components, except mRNA, were mixed via a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) to manufacture 244-9-cis lipid nanoparticles without mRNA encapsulation. 24 hours after cell seeding, the cells were treated with lipid nanoparticles in an amount of 0.5 μg, 5 μg, 50 μg, or 100 μg (based on ionizable lipids) per well. 24 hours following lipid nanoparticle treatment, Cell counting Kit—8 (Sigma-Aldrich, 96992) was added at 10 μl per well. After incubation for 4 hours, the absorbance at 450 nm was measured using an Infinite®200 PRO NanoQuant (Tecan).

The results showed no cytotoxicity in each cancer cell line up to 5 μg (FIG. 5).

Experimental Example 5: Confirmation of Hepatotoxicity of Lipid Nanoparticles

Aspartate transaminase (AST) and Alanine transaminase (ALT), which can detect the presence of diseases such as hepatocellular disease or hepatitis, are normally present in the blood at low concentrations. However, when liver cells are damaged, these transaminases are released, leading to an increase in their concentration. Alkaline phosphatase (ALP) is an enzyme in the cells lining the bile ducts of the liver that is elevated with diseases such as obstruction of the bile ducts and the cessation of bile secretion in the liver. Albumin (ALB) is produced by the liver, and albumin levels are reduced in acute hepatitis, such as cirrhosis.

To confirm hepatotoxicity, 244-9-cis lipid nanoparticles encapsulated with firefly luciferase mRNA (SEQ ID NO: 1) were manufactured and administered once to 7-week-old C57BL/6 mice at a single dose of 2 mg/kg based on mRNA. The control group was ALC-0315. 24 hours after administration, blood was collected to determine the levels of AST, ALT, ALP, and ALB (FIGS. 6 and 7). In addition, liver tissue analysis images of each experimental group are shown in FIG. 8.

As a result, there was no hepatotoxicity compared to the FDA-approved ALC-0315 lipid.

From the above description, those skilled in the art to which the present disclosure pertains will understand that the present disclosure may be embodied in other specific forms without changing the technical ideas or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure is indicated by the following claims rather than the detailed description, and should be construed as including all changes or modifications derived from the meaning and scope of the claims and equivalent concepts within the scope of the present disclosure.